An intriguing characteristic of metastatic embryonal carcinoma, a type of germ cell tumor (GCT), is that they are able to differentiate to localized teratoma, which is made up of more mature, slower growing tissue of all three embryonic lineages, ectoderm, mesoderm, and endoderm. Differentiation of embryonal carcinoma (EC) is controlled by regulatory genes that mediate permanent phenotypic change. These regulatory genes are often transcriptional regulators that activate or repress patterns of gene expression that create the phenotypic change seen during stem cell differentiation. These transcription factors can not only mediate phenotypic maturation during a particular differentiation stage of GCT, but can also regulate expression of the transcription factors that are important in the next stage of differentiation. The goal of this grant is to increase understanding how the transcription factors present in the EC cell regulate the initial lineage decisions an EC cells makes as it first differentiates. The POU homeodomain protein Oct-4 and the Forkhead Box protein we isolated, FoxD3 (previously Genesis), are transcription factors preferentially expressed in EC cells. In the normal embryonic equivalent of EC cells, embryonic stem (ES) cells, downregulation of Oct-4 during gastrulation is essential for proper lineage development. We have previously found that Oct-4 can also act as a co-repressor to prevent FoxD3 from activating the promoters of the differentiation transcription factors FoxA1 and 2. This study will investigate the role of Oct-4 and FoxD3 in EC cell differentiation decisions in three specific aims. 1) The interacting domains between Oct-4 and FoxD3 will be mapped and their function analyzed in real time proteomics. 2) The biochemical mechanism by which Oct-4 represses lineage-specific transcriptional activation will be investigated. 3) How the repressor activity of Oct-4 mediates totipotentiality versus teratoma differentiation in EC cells will be studied.